The Sertoli cells of the mammalian testis are responsible for the existence of the "blood-testis barrier." Early stages of germ cell differentiation occur in the seminiferous tubules outside of the blood-testis barrier. At a specific point in development the germ cells are moved by the Sertoli cells into the region of the seminiferous tubule within the barrier where meiosis occurs. The Sertoli cells also secrete a potassium environment within the barrier may be required for meiosis to occur. The specific tasks of this proposal are to determine the requirements of rat germ cells for meiosis, and to study the mechanisms by which rat Sertoli cells move the germ cells through the barrier at a specific stage of germ cell development. This will be accomplished by using, in a complementary manner, cell morphological and biochemical techniques to study cultured rat Sertoli and germ cells. Preliminary evidence from in vitro experiments indicates that pachytene spermatocytes can progress to at least the diakinesis stage of meiosis after several days only inculture medium with ionic composition identical to that reported to exist within the seminiferous tubule. Cultured Sertoli and germ cells will be examined by time-lapse microscopy, autoradiography and electron microscopy to determine the conditions required for meiosis. To define the mechanisms and effects of germ cell interaction with Sertoli cells the following will be examined: changes in Sertoli cell morphology at light and E.M. Levels, distribution of cytoskeletal protein (tubulin and actin) and regulatory proteins (calmodulin and cAMP-dependent protein kinase inhibitor), as revealed by indirect immunofluorescence, will be correlated with Sertoli cell adenylate cyclase activity, calcium flux and protein phophorylation. In this manner it is expected that further insight will be gained regarding mammalian meiosis, germ cell development, Sertoli-germ cell interaction, cell cytoskeletal function and non-muscle cell motility.